Clinical And Prognostic Features And Survival Analysis Of Patients With Acute Myeloid Leukemia With Mutations Of ASXL1 Or Spliceosome Gene Such As SRSF2

Part Ⅰ Clinical features and survival analysis of patients with ASXL1 gene mutation in patients with newly diagnosed non-M3 acute myeloid leukemiaObjectiveWith wide application of next-generation sequencing(NGS)with high cost and time efficiency in clinical practice,many mutated genes with clinical significance have been discovered,especially in the diagnosis,stratification and prognosis guidance of hematologic malignancies.The additional sex combs like 1(ASXL1)gene is one of the most common mutated genes in myeloid hematologic malignancies,with high mutation rate of about 5-11%in acute myeloid leukemia(AML),ASXL1 protein is a complex involved in the epigenetic regulation of gene expression.Patients with ASXL1 mutation are older,have high peripheral white blood cell count at diagnosis,and have short survival.Although the overall survival time of patients with standard induction chemotherapy regimens based on standard doses of cytarabine(Ara-C)is long,the high recurrence rate is still an urgent problem.Therefore,seeking more effective therapeutic targets and more efficient individualized diagnosis and treatment may become the way to break the situation.This article aimed to retrospectively study the relevant data of newly diagnosed AML patients with ASXL1 mutation in this hospital and analyze and discuss clinical features and survival prognosis,and provide new references for clinical diagnosis and treatment.MethodsFrom January 2016 to April 2021,256 non-M3 type(WHO type)AML patients with newly diagnosed,received NGS testing and had complete clinical data were selected and conducted retrospective analytical study.According to age,three groups were included in the study:elderly(≥60 years)(92 cases),middle-aged(45-59 years)(92 cases),and young(≤44 years)(72 cases).Clinical data and survival information were collected through outpatient cases,inpatient cases,and telephone follow-up.The primary outcomes were complete remission and minimal residual disease(MRD)after first round of treatment(CR1),overall survival(OS),progression free survival(PFS).Results1.General clinical features:In 256 patients,there were 130 males and 126 females,with a median age of 53(14-87)years.At initial diagnosis,the median peripheral blood white blood cell count was 3.5(0.08479.73)×109/L,the median proportion of bone marrow blasts was 55%(0-98%),the median proportion of peripheral blood blasts was 36%(0-98%),and the proportion of aberrant cells in nuclear cells was 34%(0-97%).Among the FAB classification,147 cases(57.42%)were M5 type.According to risk stratification by genetics,70 cases were favorable,124 cases were intermediate,and 62 cases were adverse.2.Analysis of clinical correlation:ASXL 1+patients were older than ASXL1-patients and had higher WBC at initial diagnosis(P=0.004,P=0.002).In the elderly group,WBC and the proportion of aberrant cells in nuclear cells in ASXL1+patients were higher than those in ASXL1-patients(P=0.031,P=0.009).In the young group,the WBC of ASXL1+patients was higher than that of ASXL1patients(P=0.004).3.Analysis of gene mutation characteristics:In ASXL1+patients,the proportions of aberrant cells in nuclear cells in patients with base duplication and base replacement mutations were significantly higher than that in patients with base deletion mutations(median,28%vs 17%,P=0.047;median,48%vs.17%,P=0.013).Patients with high VAF(≥40%)had higher proportion of peripheral blasts than patients with low VAF(<20%)(median,47.38%vs 21.21%,P=0.029).ASXL1 mutations often occur simultaneously with IDH2(P=0.018,r=0.34)mutations.The proportion of bone marrow blasts and aberrant cells in nuclear cells in the ASXL1 single-gene mutant group were higher than those in the composite gene mutant group(69%vs 41%,P=0.024;78%vs 23%,P=0.002).4.Efficacy and survival analysis:The median CR1 rate of ASXL1+patients in the middle-aged group was significantly lower than that of ASXL1-patients(P=0.019),and the median MRD level of ASXL1+patients in the elderly and middle-aged group after the first course of chemotherapy were higher than that of ASXL1-patients(P<0.001,P=0.005).The use of different induction regimens had significant effect on the CR1 rate,and the CR1 rate of IA regimen was higher than that of DA regimen in ASXL1-patients in the youth group(P=0.002).The median OS time of ASXL1+and ASXL1-patients were 10 months and 20 months(P=0.012),and the median PFS time were 10 months and 17 months,respectively(P=0.002).In the elderly group,there was no significant difference in OS time between ASXL1+and ASXL1-patients(P>0.05),while the median PFS time of ASXL1+patients was 5 months,which was significantly shorter than that of ASXL1-patients(P=0.029).In the middle-aged group,the OS time of ASXL1+and ASXL1-patients were 11 months and 19 months(P=0.033),and the PFS time were 10 months and 16 months,respectively(P=0.047).In the young group,the OS time of ASXL1+and ASXL1-patients were 28 months and 37 months(P=0.620)，and the PFS time were 28 months and 36 months,respectively(P=0.520).5.Prognosis analysis:The factors with P<0.05 in univariate analysis(proportion of aberrant cells in nuclear cells,complex karyotype and TET2 mutation)were included in the Cox multivariate analysis.The results showed that the proportion of aberrant cells in nuclear cells(HR=2.678,P=0.020),complex karyotype(HR=6.824,P=0.032)and TET2 mutation(HR=2.441,P=0.041)were independent risk factors affecting OS in ASXL1+patients.Conclusion1.ASXL1 mutant non-M3 AML patients had higher age,higher peripheral blood WBC and higher aberrant cells in nuclear cells at initial diagnosis.2.Base duplication mutation,base substitution mutation and high VAF of ASXL1 mutation may be related to poor prognosis.3.AML patients with ASXL1 mutation had shorter OS and PFS time,lower CR rate after the first course of chemotherapy,higher MRD levels,and poor overall survival outcomes.4.The proportion of aberrant cells in nuclear cells,complex karyotype and TET2 mutation were independent risk factors affecting the prognosis of AML patients with ASXL1 mutation.Part Ⅱ Clinical and prognostic characteristics and pathogenesis of patients with SRSF2 and related spliceosome gene mutations in newly diagnosed acute myeloid leukemia patientsObjectiveMutations in the splicing factor(SF)gene are often detected in myelodysplastic syndromes(MDS),but the clinical and prognostic correlation of these mutations in acute myeloid leukemia(AML)has rarely been reported.About 10%-18%of are manifested as spliceosome gene mutation in the gene mutation spectrum of acute myeloid leukemia.And the survival outcomes of these patients are poor,similar in treatment to most patients with acute myeloid leukemia,but there is a lack of safe and effective targeted drug intervention.This study aimed to retrospectively study the relevant data of patients with SF gene mutation in this hospital who are newly diagnosed with AML and analyze their clinical features and prognosis,find mutation sites that may become new targets,and provide new ideas for the diagnosis and treatment of such diseases.MethodsAccording to the 2016 WHO diagnostic typing criteria,we collected 368 patients with non-M3 AML who were initially diagnosed from 2015 to 2021 in this hospital.Four spliceosome genes were detected using next-generation sequencing(NGS):serine/arginine rich splicing factor 2(SRSF2),U2 small nuclear ribonucleoprotein auxiliary factor 35kD subunit-related protein 2(ZRSR2),splicing factor 3b subunit 1(SF3B1),and U2 small nuclear RNA auxiliary factor 1(U2AF1).Along with 34 other genes,a total of 38 gene were detected.According to whether they were accompanied by SF gene mutations,368 patients were divided into SF-mutated group and SF-wild group,and further divided SF mutation patients into four groups according to mutated genes.And the clinical characteristics such as blood routine and blast proportion,as well as the treatment outcomes such as complete remission(CR)rate after the first course of treatment,the level of minimal residual disease(MRD)after the first course of treatment,and the overall survival(overall)were compared.Finally,the function of SRSF2P95H mutant cells was detected using cell counting kit 8(CCK8).Results1.Analysis of clinical features:In 368 patients,there were 193 male and 175 female,with a median age of 53(14-87)years.The median WBC count was 8.35(0.08-479.73)×109/L,the median proportion of bone marrow blasts was 40%(0-98%),and the median proportion of aberrant cells in nuclear cells was 63%(0-98%)at initial diagnosis.Among the FAB classification,223 cases(60.60%)were M5 type.According to risk stratification by genetics,339 patients were classified:106 cases were favorable,126 cases were intermediate,and 107 cases were adverse.2.Analysis of mutation characteristics:SF mutations accounted for 17.4%(64/368)of all patients,while SRSF2,U2AF1,SF3B1 and ZRSR2 mutations accounted for 51.5%,29.7%,14.1%and 4.7%,respectively.Mutation of SRSF2 mainly occurred in the 95th position with proline replaced by histidine.Correlation analysis showed that SRSF2 was often co-mutated with IDH2 and NPM1(r=0.47,r=0.33,P<0.05),but negatively correlated with U2AF1 mutation(r=-0.33,P<0.05).3.Comparison of clinical features:3.1 SF-mutated and SF-wild patients:SF mutations were more common in older and male patients compared with SF-wild patients(median,64 vs 52 years;0.750 vs 0.477,P<0.001).In SF-wild patients,the proportion of patients with adverse risk stratification by genetics was significantly lower than that of moderate and good patients(0.24 vs 0.31 or 0.37,P<0.05),while the proportion was significantly higher in SF-mutated patients(0.50 vs 0.22 or 0.19,P<0.001).3.2 SRSF2-mutated and SF-wild patients:Compared with SF-wild patients,the age,proportion of peripheral blasts,and the proportion of aberrant cells in nuclear cells were higher in SRSF2-mutated patients at initial diagnosis(64 vs 61 years old,P=0.015;0.48 vs 0.14,P=0.028;0.48 vs 0.23,P=0.014).3.3 SRSF2P95H and other SRSF2-mutated patients:Compared with other SRSF2-mutated patients,SRSF2P95H patients had higher proportion of peripheral blasts and aberrant cells in nuclear cells at initial diagnosis(0.77 vs 0.32,P=0.031;0.79 vs 0.23,P=0.026).4.Analysis of Efficacy and survival:Efficacy analysis:Compared with the SF-wild group,the level of minimal residual disease after the first course of chemotherapy was higher than that in SF-mutated group,and the complete response rate after the first course of chemotherapy was lower(0.035 vs 0.008,P=0.018;0.48 vs 0.75,P<0.001).Survival analysis:The median overall survival time of SF-mutated patients was shorter than that of SF-wild patients(17 months vs 24 months,P=0.041).Moreover,the median overall survival time of SRSF2-mutated patients tended to be shorter than that of SF-wild patients(13 months vs 24 months,P=0.064).There was also a trend towards shorter median overall survival time in patients with secondary AML than in newly diagnosed AML(13 months vs 22 months,P=0.227).There were significant differences in median overall survival time(10 months,22 months,and 31 months,P=0.027)in patients with three mutation types(SRSF2mutIDH2wt,SRSF2mutIDH2R140Q and SRSF2wtIDH2R140Q).5.Prognosis analysis:The factors with P<0.05 in univariate analysis(male,ASXL1 mutation,Kit mutation and ZRSR2 mutation)were included in multivariate Cox analysis,and the results showed that male,Kit mutation and ZRSR2 mutation were independent risk factors affecting the overall survival of patients.6.Analysis of SRSF2P95H function:The proliferation of SRSF2P95H and SRSF2wt cells was detected at 24h,48h,72h and 96h,respectively,and the results showed that the former significantly promoted the proliferation of Molm13 cells after 72 and 96h(P<0.05).Conclusion1.SRSF2 mutation accounted for the highest proportion of spliceosome mutations,and SRSF2 mutation mainly occurred in P95H.SRSF2 was often co-mutated with IDH2 and NPM1,and negatively correlated with U2AF1 mutation.2.SF mutations were often associated with high age,male and adverse risk stratification by genetics.SRSF2 mutation,especially the P95H site,was often associated with higher proportion of peripheral blasts and aberrant cells in nuclear-cells.3.SF-mutated patients had higher MRD and lower CR rate after the first course of chemotherapy and had shorter overall survival time.Patients with SRSF2mutDH2wt mutations showed poor survival outcomes.4.Male,Kit mutation and ZRSR2 mutation were independent risk factors affecting the prognosis of SF-mutated AML patients.5.SRSF2P95H promoted the proliferation of leukemia molm13 cells.